Name: isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation
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Diese Arbeit wurde unterstützt durch einen Zuschuss aus den Natur- und Ingenieurwissenschaften Research Council of Canada unterstützt (NSERC, gewähren keine 298.515-2011). AL ist der Empfänger eines Ph.D. Stipendium von den Natur- und Ingenieurwissenschaften Research Council of Canada (NSERC) und MS wurde aus einem Zuschuss von der kanadischen Institutes of Health Research (CIHR, gewähren keine. MOP123246).

Alle Verfahren wurden in Übereinstimmung mit dem Canadian Council on Animal Care Leitlinien nach Genehmigung durch die Institutional Animal Care Committee des Centre de recherche du Centre Hospitalier de l&#39;Université de Montréal (CRCHUM) durchgeführt. 1. Isolierung, Identifizierung und Kultur von Murine Peritonealmakrophagen <ol><li> Bereiten Sie 3,8% Brau Thioglykolat-Medium. Um dies zu tun, aussetzen 38 g Thioglykolat-Medium in 1000 ml destilliertem Wasser. Bringen Lösung zum Kochen bringen, um das Medium vollständig zu lösen. Sterilisieren durch Autoklavieren bei 121 ° C für 15 min. Speichern Sie bis zu 3 Monate in der Dunkelheit, bei RT 18. HINWEIS: Verwerfen, wenn Trübung entwickelt, die eine bakterielle Kontamination zeigt. Die Lösung kann bis zu einem Jahr im Dunkeln gehalten werden, wenn steril gehalten. </li><li> Verwendung von 1 ml-Spritzen bis 23 G Nadeln befestigt injizieren 1 ml 3,8% Brewer Thioglykolat-Medium in die Peritonealhöhle jeder Vereinbarungse und warten Sie für 3 Tage. Verwenden Sie neue Spritze und Nadel für jede Maus. </li><li> Betäuben Mäusen durch intraperitoneale Injektion von Natriumpentobarbital (100 mg / kg) und euthanize Mäuse durch Genickbruch. Überprüfen Sie die ordnungsgemäße Betäubung durch Überprüfung der Atemfrequenz. In der Regel schnelle Atmung zeigen, dass die Maus nicht tief anästhesiert. </li><li> Den Unterleib jeder Maus mit 70% Ethanol waschen. Mit einer Schere, führen Sie einen seitlichen Schnitt entlang der unteren Mittellinie des Bauchfells. </li><li> Mit einer Pinzette, ziehen Sie die Bauchhaut, um die transparente Bauchhaut freizulegen. Verwendung von 5 ml Spritzen an 20 G Nadeln befestigt injizieren 5 ml kaltem DPBS in die Peritonealhöhle jeder Maus. </li><li> Führen Sie eine sanfte Massage auf der Bauchhöhle und dann sorgfältig absaugen Flüssigkeit ohne Punktieren jedes Organ. Entfernen Sie die Nadel und verzichtet der Peritonealflüssigkeit in 50 ml konischen Zentrifugenröhrchen. </li><li> Zentrifugieren für 10 min bei 400 × g in einer gekühlten Zentrifuge.Zellen müssen während des gesamten Verfahrens kalt bleiben. Überstand verwerfen und resuspendieren Zellpellet in RPMI-Medium 1640. </li><li> Verwendung eines Hämozytometers zählen Zellen und einzustellen, um die Zelldichte auf 1 x 10 6 Zellen / ml. </li><li> Charakterisierung des Phänotyps isolierten Zellen durch Durchflusszytometrie unter Verwendung von 1 x 10 6 Zellen pro Maus und antibodie gegen F4 / 80 (eine Oberfläche Antigens, das auf Makrophagen). </li></ol> 2. Zellbehandlungen <ol><li> Direkt nach der Isolierung, fügen Sie 1 x 10 6 Zellen in jede Vertiefung. Lassen die murine peritoneale Makrophagen durch Kultivieren für 1 bis 2 Stunden bei 37 ° C in der 6-Well-Platten zu halten. Entfernen Sie nicht anhaftende Zellen durch vorsichtiges Waschen 3 mal mit warmem PBS. </li><li> Anschließend Kulturzellen in 900 &amp; mgr; l serumfreiem DMEM für 24 h in Gegenwart der folgenden TLR-Liganden: (Pam3CSK4-TLR1 / 2 (0,5 mg / ml), Poly (I: C) -TLR3 (10 mg / ml) ; LPS-TLR4 (100 ng / ml); Flagellin-TLR5 (100 ng / ml); FSL1- TLR6 / 2 (100 ng / ml); ssRNA40-TLR7 (1 ug / ml); ODN1826-TLR9 (1 uM). HINWEIS: Bereiten Sie für jeden Liganden ein 10-faches Konzentrat. 100 l des letzteren in jede Vertiefung, wodurch die Durchführung einer 10-fachen Verdünnung. </li></ol> 3. RNA-Isolierung <ol><li> Entfernen Sie alle mittleren und lysieren Zellen direkt in den sechs-Well-Platten durch Zugabe von 1 ml TRIzol in jede Vertiefung und das Bestehen der Zelllysat mehrmals in eine Pipette. Die homogenisierte Proben inkubieren 5 bis 10 Minuten bei Raumtemperatur, um die vollständige Dissoziation Nukleoproteinkomplexen ermöglichen. </li><li> Übertragen Lysat in 1,5 ml RNase- und DNase-freie Mikrozentrifugenröhrchen. 0,2 ml Chloroform pro 1 ml TRIzol. Durch kräftiges Schütteln Rohre von Hand für 15 Sekunden und inkubieren bei Raumtemperatur für 5 bis 10 min. Zentrifuge bei 12.000 xg für 15 min bei 4 ° C. Beachten Sie die Mischung getrennt wird in eine untere rote Phase (Phenol-Chloroform), eine Interphase und eine farblose obere wässrige Phase. RNA liegt ausschließlich in der wässrigen Phase. </li><li> Transfer sorgfältig die obere wässrige Phase in ein frisches Röhrchen, ohne die Interphase zu stören. Ausfällung der RNA aus es durch Mischen mit 0,5 ml Isopropylalkohol. Proben Inkubation bei RT für 10 Minuten und Zentrifugation bei 12.000 × g für 10 min bei 4 ° C. Die RNA-Ausscheidungen bildet ein weißes Pellet an der Seite und am Boden des Röhrchens. </li><li> Überstand entfernen. Mit 1 ml 75% Ethanol waschen das RNA-Pellet einmal. Mischen Sie die Proben durch Vortexen und Zentrifugieren bei 7.500 xg für 5 Minuten bei 4 ° C. Kurz trocknen die RNA (Luft trocknen für 10 min). Lösen sich RNA in 0,1 ml DEPC (RNAse freies Wasser) und Inkubation für 10 min bei 55 ° C. </li><li> Quantifizieren RNA mit einem Spektralphotometer. Um dies zu tun, verdünnte Proben 100 Mal in DEPC Wasser und bei Wellenlängen von 260 nm gelesen. Die RNA-Konzentration ist dann: OD 260 x 40 ng / ul x Verdünnungsfaktor. </li><li> RNA-Konzentrationen auszugleichen und zu synthetisieren cDNA gemäß den Anweisungen des Herstellers unter Verwendung von RT-PCR-System für First-Strand cDNA Synthesis Kit. Wieden Anweisungen des Herstellers, messen Gen-mRNA-Spiegel durch Echtzeit-PCR (45 Zyklen) in einer Echtzeit-DNA-Detektionssystem. Normalisierung der Genexpression Ebenen mit zwei Housekeeping-Gen beispielsweise β-Actin und GAPDH. HINWEIS: Normalisieren RNA-Konzentration auf 500 ug / ml und mit 0,5 &amp; mgr; g für die cDNA-Synthese. </li></ol>

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The authors have no competing financial interests.

Makrophagen sind von entscheidender Bedeutung für das Überleben und ein verlockendes Ziel, um den Host für immunologische Ziele zu manipulieren. Die Entdeckung von TLR und andere Erkennungsmoleküle das Makrophagen zur Mitte der immunologischen Debatte geführt. Makrophagen sind auf eine Vielzahl von Stimuli, einschließlich Cytokinen, Schadens-associated molecular pattern Moleküle (dämpft) 20 und Moleküle mit Gruppen von Pathogenen (PAMPS) 21 verbunden. Diese Reaktionen unterschiedliche Stim...

Retikuloendothelialen phagozytische System von Zellen in verschiedenen Geweben und Organen, wie Knochenmark, Blut, Leber und Milz besteht. Makrophagen werden in großem Umfang um den Körper, wo sie vor allem in angeborenen teilzunehmen und adaptiven Immunantwort zu steuern und zu Infektionen klar verteilt. Zusätzlich zu ihrer Rolle bei der Immunabwehr, Makrophagen spielen eine wichtige Rolle bei der Wundheilung und bei der Aufrechterhaltung Gewebshomöostase 1,2. Darüber hinaus sind Makrophagen nicht nur die Immunfunktion wichtig, sondern auch aktiv in Eisenhomöostase 3 teilzunehmen. Im Körper ist etwa 80% der Eisen im Hämoglobin in Erythrozyten vorhanden ist, die bei seneszenten werden von Makrophagen phagozytiert 4. Täglich, diese Makrophagen recyceln 25 mg von Erythrozyten-abgeleitete Eisen und bringt ihren Transport in das Plasma 5. Darüber hinaus während der Infektion und Entzündung, proinflammatorischen Makrophagen maskieren Serum-Eisen auf Eisen VERFÜGBARK reduzierenty, um Krankheitserreger, die an beiden systemischen und lokalen Ebenen 6-8. Wie gut, Studien haben gezeigt, dass vor allem Makrophagen und Hepatozyten erzeugen ein antimikrobielles Peptid namens Hepcidin, das als das Master-Regulator des Eisenstoffwechsels 9, 10 ist. Hepcidin wird hauptsächlich durch inflammatorische Stimuli erhöht und ist teilweise für die Eisenspeicherung in Makrophagen bei chronischen Entzündungen 11-13 verantwortlich. Wie Hepcidin-Expression in Makrophagen ist nicht sehr gut verstanden, haben wir untersucht die mögliche Rolle von Toll-like Rezeptoren (TLRs) in dieser Regelung. Die TLRs sind in erster Linie auf Makrophagen und spielen eine zentrale Rolle in der Aktivierung. Zusätzlich ist LPS induzierte Hepcidin Expression in der Leber hängt von TLR4 13. Daher unserer Untersuchung auszuführen, verwendeten wir ein Verfahren, das auf die Isolierung von murinen peritonealen Makrophagen. Makrophagen-Zelllinien werden im Großen und Ganzen in Makrophagen Zucht eingesetzter Jahren; dennoch ausgedehnte Kultur Genverluste provozieren und verringerte Immunfunktionen in diesen Zelllinien. Somit ist die Isolierung von Makrophagen aus der Peritonealhöhle entscheidend. Die Maus Bauchhöhle stellt eine ideale Ort, um Makrophagen 13-15 ernten. Isolated murinen peritonealen Makrophagen sind bequem für mehrere Studien in Bezug auf ihre immunologische Funktion. Jedoch ist die Anzahl von Makrophagen in das Peritoneum nicht ausreichend ist für umfangreiche Untersuchungen und wird geschätzt etwa 1 x 10 6 Makrophagen pro Maus. Somit Makrophagen Ausgang zu erhöhen, wurde eine sterile Hervorrufen Mittel wie Thioglykolat in die Bauchhöhle vor der Zellernte injiziert. Nach Thioglycolat Injektion wurde die Ausbeute an Makrophagen pro Maus durch das 10-fache erhöht. Trotz der Zunahme der Makrophagen Ertrag, Thioglycolat-Medium wirkt Brewer als Reizmittel, die eine Entzündungsreaktion induziert, was zur Rekrutierung von Makrophagen, die may, aber nicht notwendig Auswirkungen auf die Genexpression. Daher muss eine Kontrollgruppe, bestehend aus nicht-behandelten Makrophagen in jedem Experiment eingeschlossen sein. In unseren Händen, wurde Hepcidin-Expression, die stark durch eine Entzündung stimuliert wird nicht in nicht-behandelten Thioglycolat erkannt ausgelöst Peritonealmakrophagen. Darüber hinaus haben Studien gezeigt, dass Brewer Thioglycolat rekrutiert zahlreiche Makrophagen, aber nicht 16 aktivieren. Andererseits löste Brewer Thioglycolat Makrophagen zeigten einen Anstieg der lysosomalen Enzyms, aber eine Verringerung in der Tötung von Mikroorganismen 17 eingenommen. Allerdings wurde die Phagozytose nicht, wenn sie mit nicht-ausgelöst Makrophagen 16 im Vergleich betroffen. Einmal in Schalen kultiviert, die Peritonealmakrophagen verwachsen und ermöglicht damit ihre Trennung von einer anderen Art von Zellen, die aus der Peritonealhöhle isoliert wird. Anschließend wurden die isolierten Makrophagen mit verschiedenen TLR Agonisten herausgefordert.Schließlich mRNA wurde aus den kultivierten Zellen extrahiert, und die Genexpression wurde durch quantitative reverse Transkriptase-Polymerasekettenreaktion (qRT-PCR) analysiert.

<table><tbody><tr><td>C57BL/6 mice</td><td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td><td>475</td><td></td></tr><tr><td>Thioglycollate</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>19032-500G</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td><td>WISENT INC Canada (QC)</td><td>311-425-CL</td><td></td></tr><tr><td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td><td>WISENT INC Canada (QC)</td><td>350-000-CL</td><td></td></tr><tr><td>1 and 5 ml syringes</td><td>BD USA (NJ)</td><td>309659</td><td></td></tr><tr><td>6-well plates</td><td>Corning Incorporated (NY, USA)</td><td>MCT-150-C</td><td></td></tr><tr><td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-pm25</td><td></td></tr><tr><td>Polyionosine&amp;ndash;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-PIC</td><td></td></tr><tr><td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>L2880</td><td></td></tr><tr><td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FLIC-10</td><td></td></tr><tr><td>Lipoprotein synthetic FSL1 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FSL</td><td></td></tr><tr><td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &amp;mu;g/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-LRNA-40</td><td></td></tr><tr><td>Type B CpG oligonucleotide ODN1826 (1 &amp;mu;M)</td><td>InvivoGen (San Diego, USA)</td><td>11B16-MM</td><td></td></tr><tr><td>TRIzol</td><td>Invitrogen, (Burlington, ON, Canada)</td><td>15596-026</td><td></td></tr><tr><td>20 G and 23 G needles</td><td>BD USA (NJ)</td><td>305175</td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tubes placed on ice</td><td>Sarstedt (Newton, MA, USA)</td><td>62.547.205</td><td></td></tr><tr><td>Red Blood Cells Lysis Buffer</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>R7757-100ML</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>F4/80 antibody</td><td>BIO-RAD ( CA, USA)</td><td>MCA497APC</td><td></td></tr><tr><td>CD16/CD32 antibodies</td><td>Pharmingen, {Mississauga, ON, CA)</td><td>553141</td><td></td></tr><tr><td>Flow cytometer</td><td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Axygen Scietific (CA,USA)</td><td>3516</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific (ON, Canada)</td><td>UN1888</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>JT Baker (PA, USA)</td><td>70566</td><td></td></tr><tr><td>75% ethanol (in DEPC treated water)</td><td>Commercial Alchohols (QC, Canada)</td><td>17394</td><td></td></tr><tr><td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>216.542.8</td><td></td></tr><tr><td>Omniscript RT-PCR system</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>205113</td><td></td></tr><tr><td>Rotor Gene 3000</td><td>Montreal Biotech, (Kirkland, QC, Canada)</td><td></td><td></td></tr><tr><td>QuantiTect SYBR Green I PCR kits</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>204141</td><td></td></tr></tbody></table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

Während der Infektion und Entzündung, lassen zirkulierenden Monozyten in die Blutbahn und wandern ins Gewebe, wo sie in Makrophagen zu differenzieren. Makrophagen exprimieren Oberflächen Toll-like Rezeptoren (TLRs), die molekulare Muster in einer Vielzahl von Mikroorganismen durch Evolution konserviert zu erkennen. TLRs spielen eine zentrale Rolle in Makrophagenaktivierung, die in der Regel mit Genexpressionsänderung verbunden ist. Makrophagen sind kritisch bei vielen Krankheiten und werden als attraktive Ziele für eine Therapie entwickelt. In dem folgenden Protokoll beschreiben wir ein Verfahren, um murine peritoneale Makrophagen mit Brauerei Thioglykolat-Medium zu isolieren. Letzteres wird Monozytenmigration in das Peritoneum zu steigern, entsprechend wird dies Makrophagen-Ausbeute um das 10-fache zu erhöhen. Mehrere Studien wurden unter Verwendung von Knochenmark, Milz und peritoneale Makrophagen durchgeführt. Allerdings wurden peritoneale Makrophagen gezeigt reiferen auf Isolation zu sein und sind in ihrer funktionalen stabilerkeit und Phänotyp. So Makrophagen aus murinen Bauchhöhle isoliert präsentieren eine wichtige Zellpopulation, die in verschiedenen immunologischen und metabolischen Untersuchungen dienen kann. Nach der Isolierung wurden Makrophagen mit verschiedenen TLR-Liganden stimuliert und damit die Genexpression wurde ausgewertet.

Zunächst gekennzeichnet die isolierten murinen peritonealen Makrophagen mittels Durchflusszytometrie. Um dies zu tun, haben wir (F4 / 80) Antikörper, die spezifisch Marker nur durch Makrophagen zu erkennen. Diese Charakterisierung wird benötigt, um die Prozentzahl der isolierten Makrophagen zu bestimmen und sie unter den Zellen während des Isolierungsprozesses erhaltenen unterscheiden. Wie in (1) gezeigt, ist der Prozentsatz von Zellen, die das Antigen F4 / 80 wurde übereinstimmend festgestellt, ü...

Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolierung von Murine Peritonealmakrophagen zur Durchführung Genexpressionsanalyse Nach Toll-like Rezeptoren Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

isolation-murine-peritoneal-macrophages-to-carry-out-gene-expression

Research

JoVE Journal

Immunology and Infection

36.2K Aufrufe.  Institut du cancer de Montréal. We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Serie: Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Isolierung von Maus Bauchhöhle Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Forschung

Immunologie und Infektion

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Eine einfache und effiziente Methode, um Makrophagen aus Mixed primären Kulturen von adulten Leber Zellen zu isolieren

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Untersuchung der Auswirkungen von tumorgeheimen Parakrinliganden auf die Makrophagenaktivierung mit Co-Culture mit permeableMembranen unterstützt

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Krebsforschung

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Mit RNA-Interferenz, um die angeborene Immunantwort in Makrophagen der Maus Untersuchen

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolierung humaner Monozyten durch Doppel-Zentrifugation und ihre Differenzierung zu Makrophagen in Teflon-beschichteten Zellkultur-Taschen

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Bewertung der Antikörper-basierte Medikamente Auswirkungen auf murinen Knochenmark und peritonealen Makrophagen Aktivierung

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Screening bioaktiver Nanopartikel in phagozytischen Immunzellen für Inhibitoren der Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Medizin

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Experimentelle Analyse des Apoptotic Thymozyte Engulfment durch Macrophagen

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Isolierung und Kultivierung von Makrophagen aus dem Knochenmark aus Mäusen

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Source: Zhen, Y. et al., <a href="https://app.jove.com/v/59731">Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

Analyse der Efferozytose apoptotischer Thymozyten mittels Peritonealmakrophagen

Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure ...